The main objective of this research is to select out and characterize early and late stages in the evolution of neoplasia in the respiratory epithelium. To do this effectively, it is necessary to be able to separate out discrete cell populations identified as particular stages in this sequence of events and to follow the progression of neoplasia in the cell populations evolving from the original cells. An in vivo-in vitro approach is used to identify, in a nondestructive manner, the lesions on pieces of DMBA- preexposed rat tracheal implants from the cytopathology in the cells which exfoliate from these pieces in organ culture. Cell populations are then generated directed from the pieces as outgrowths of epithelial cells in primary culture and markers of the progression of neoplasia are looked for in these cell cultures and the cell lines established from them. The plan is to use this approach to continue to investigate the cellular and biochemical properties of the early to late events in the progression of neoplasia in respiratory epithelium and to correlate morphological markers with these properties. To carry this out, the first aim is to continue to quantitate the effects of DMBA exposure conditions that will induce large numbers of carcinogen-altered cell populations that are in early stages, and ones that are synchronized by repeat exposure to the late stages of carcinogenesis. These carcinogen-altered cell populations are separated out from normal cells by the selective survival of the altered cells in pyruvate-deprived medium. The second aim is to investigate the underlying mechanisms which permit survival of carcinogen-altered cells in the pyruvate-deprived medium. To do this, the differences in utilization of key metabolites for biosynthetic pathways and energy production and enzyme activities related to pertinent pathways will continue to be studied in the normal, carcinogen-altered and tumorigenic cell population. In particular, the control of expression of mitochondrial-linked malic enzyme activity, which is markedly elevated in carcinogen-altered cells, will be studied. The third aim is to continue to investigate the progression of in vivo markers of neoplasia, and how these functional markers arise within the evolving cell populations. Obtaining more synchronous cell populations as described in the first specific aim should help to correlate functional markers with morphological and biochemical markers of stages in the progression of neoplasia.